U.S. patent application number 15/478290 was filed with the patent office on 2017-08-31 for methods of nucleic acid fractionation and detection.
The applicant listed for this patent is Meridian Bioscience, Inc.. Invention is credited to Vecheslav A. Elagin, Anand Hindupur, Ahmer Kodvawala, Brian Loeffler, Reddy Ponaka, Vladimir I. Slepnev.
Application Number | 20170247680 15/478290 |
Document ID | / |
Family ID | 49624537 |
Filed Date | 2017-08-31 |
United States Patent
Application |
20170247680 |
Kind Code |
A1 |
Slepnev; Vladimir I. ; et
al. |
August 31, 2017 |
Methods of Nucleic Acid Fractionation and Detection
Abstract
The invention provides methods of detecting a nucleic acid
present in a biological sample, comprising combining the biological
sample with a lysis buffer to form a lysis mixture comprising
nucleic acid released from cells in said biological sample; and
subjecting a volume of the lysis mixture to size-exclusion
chromatography in a column comprising a volume of size-exclusion
medium. In certain embodiments, the lysis buffer separates
double-stranded nucleic acid into single-stranded nucleic acid. In
certain embodiments, the elution can have a flow rate of separation
of less than 10 minutes to produce an eluted solution comprising
isolated nucleic acid. The invention provides for a method of
accurately and rapidly detecting products of nucleic acid
amplification.
Inventors: |
Slepnev; Vladimir I.;
(Cincinnati, OH) ; Kodvawala; Ahmer; (Cincinnati,
OH) ; Loeffler; Brian; (Cincinnati, OH) ;
Hindupur; Anand; (Cincinnati, OH) ; Ponaka;
Reddy; (Cincinnati, OH) ; Elagin; Vecheslav A.;
(Cincinnati, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Meridian Bioscience, Inc. |
Cincinnati |
OH |
US |
|
|
Family ID: |
49624537 |
Appl. No.: |
15/478290 |
Filed: |
April 4, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14546478 |
Nov 18, 2014 |
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15478290 |
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PCT/US2013/042671 |
May 24, 2013 |
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14546478 |
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61651426 |
May 24, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C12Q 1/6806 20130101; C12N 15/101 20130101; C12Q 2527/137 20130101;
C12Q 2527/125 20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10; C12Q 1/68 20060101 C12Q001/68 |
Claims
1.-21. (canceled)
22. A method of purifying a nucleic acid from a biological sample,
comprising the steps of: a) combining the biological sample with a
lysis buffer to form a lysis mixture comprising nucleic acid
released from cells in said biological sample; b) applying a volume
of the lysis mixture to size-exclusion chromatography medium in a
column comprising a loading end, an eluting end, and a volume of
size-exclusion medium, wherein said volume of lysis mixture is 0.35
to 0.8 of the volume of the size-exclusion medium, and allowing
lysis mixture to completely enter the size-exclusion chromatography
medium; and c) providing a positive pressure differential to the
loading side of the column forcing interstitial fluid containing
nucleic acid from to drain from SEC medium, and collecting drained
fluid containing nucleic acid for further amplification or
analysis.
23. The method of claim 22, wherein the lysis mixture enters the
SEC medium by gravity flow.
24. The method of claim 22, wherein said nucleic acid is DNA, RNA,
or a mixture thereof.
25. The method of claim 22, further comprising equilibrating said
chromatography column with an equilibrating buffer comprising 1-10
mM Mg.sup.2+ or a non-ionic detergent, or a combination, prior to
the subjecting of step b).
26. The method of claim 22, wherein said lysis buffer comprises
alkali hydroxide at pH>11.
27. The method of claim 22, wherein said lysis buffer comprises
urea or chaotropic salt.
28. The method of claim 22, wherein said lysis buffer separates
double stranded nucleic acid into single stranded nucleic acid and
inhibits nucleic acid interactions with protein.
29. The method of claim 22, wherein said size-exclusion medium
comprises polyacrylamide, polybisacrylamide or
polymethacrylamide.
30. The method of claim 22, wherein said nucleic acid is DNA, RNA,
or a mixture thereof.
31. The method of claim 22, further comprising the later step of
analyzing said isolated nucleic acid using an enzyme-catalyzed
reaction.
32-43. (canceled)
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation application of PCT
Application No. PCT/US2013/042671 filed on May 24, 2013 which
claims the benefit of U.S. Provisional Patent Application No.
61/651,426, filed May 24, 2012, which are hereby incorporated by
reference in their entirety.
FIELD OF INVENTION
[0002] The present invention relates to methods of isolation and
purification of nucleic acids from a biological sample or matrix.
More particularly, the present invention relates to the isolation
and purification of nucleic acids using size-exclusion or gel
filtration chromatography.
BACKGROUND OF THE INVENTION
[0003] The isolation and identification of nucleic acids are
important steps in many biochemical detection and clinical
diagnostic tests. The separation of nucleic acids from the complex
cellular compositions in which they are found is often a necessary
initial step before detection or amplification can be undertaken.
The presence of large amounts of cellular debris, such as proteins
and carbohydrates, in the compositions often impedes the reactions
and techniques used in molecular biology. The presence of exogenous
agents frequently used for nucleic acid isolation can also inhibit
nucleic acid amplification. Therefore, the current isolation and
amplification procedures are undesirably time consuming,
complicated, and inefficient. Thus, improved methods for the
isolation and detection of nucleic acids, are desirable, for a
broad variety of applications in medical diagnostics for microbial
infections, detection of genetic variations, forensic science,
tissue and blood typing, detection of environmental pathogens, and
basic research, to name a few.
[0004] A range of methods are known for the isolation and
purification of nucleic acids, but generally, these rely on a
complex series of extraction and washing steps and are time
consuming and laborious to perform. Classical methods for the
isolation of nucleic acids from complex starting materials, such as
blood, blood products, tissues, or other biological materials,
involve lysis of the biological material, followed by isolation
strategies such as solid phase extraction or phenol extraction
followed with ethanol precipitation.
[0005] Paramagnetic bead technology has also been used for nucleic
acid isolation. Most magnetic bead-based methods rely on lysing the
sample followed by binding nucleic acids with magnetic beads and
washing. Isolated nucleic acids obtained from these methods often
contain agents, such as ethanol, which inhibit further
amplification and detection.
[0006] Size-exclusion chromatography (SEC), also called
gel-filtration or gel-permeation chromatography (GPC), uses porous
particles stacked within a column to separate molecules of
different sizes. It is generally used to separate biological
molecules, and to determine molecular weights and molecular weight
distributions of polymers. Molecules that are smaller than the pore
size can enter the particles and therefore have a longer path and
longer transit time than larger molecules that cannot enter the
particles. Molecules larger than the pore size can not easily enter
the pores, and elute together earlier in the chromatogram.
Molecules that can enter the pores have an average residence time
in the particles that depends on the molecular size and shape.
Different molecules therefore have different total transit times
through the column.
[0007] There is still a need for improved nucleic acid purification
and detection methods which are quick, economical and simple to
perform, which enable detectable yields to be obtained with minimal
losses, whereby the nucleic acids obtained are ready for downstream
amplification and analysis.
SUMMARY OF THE INVENTION
[0008] In certain embodiments, the invention provides a method of
detecting a nucleic acid present in a biological sample, comprising
the steps of: a) combining the biological sample with a lysis
buffer to form a lysis mixture comprising nucleic acid released
from cells in said biological sample; b) subjecting a volume of the
lysis mixture to size-exclusion chromatography in a column
comprising a volume of size-exclusion medium, wherein said volume
of lysis mixture is 0.01 to 0.6 of the volume of the size-exclusion
medium, and having a flow rate of separation of less than 10
minutes to produce an eluted solution comprising isolated nucleic
acid; c) combining the eluted solution with nucleic acid
amplification reagents comprising DNA polymerase, oligonucleotides
and nucleoside triphosphates for nucleic acid amplification; d)
amplifying the nucleic acid in the eluted solution; and e)
detecting products of nucleic acid amplification.
[0009] The present invention also provides a method of purifying a
nucleic acid from a biological sample, comprising the steps of: a)
combining the biological sample with a lysis buffer to form a lysis
mixture comprising nucleic acid released from cells in said
biological sample; and b) subjecting a volume of the lysis mixture
to size-exclusion chromatography in a column comprising a volume of
size-exclusion medium, wherein said volume of lysis mixture is 0.01
to 0.6 of the volume of the size-exclusion medium, and having a
flow rate of separation of less than 10 minutes to produce an
eluted solution comprising isolated nucleic acid. In certain
embodiments, the invention provides the later step of analyzing
said isolated nucleic acid using an enzyme-catalyzed reaction.
[0010] The present invention further provides a method of purifying
a nucleic acid from a biological sample, comprising the steps of:
a) combining the biological sample with a denaturing solution that
separates strands of double-stranded DNA; b) subjecting a volume of
the biological sample mixture to size-exclusion chromatography in a
column comprising a volume of size-exclusion medium, wherein said
volume of lysis mixture is 0.01 to 0.6 of the volume of the
size-exclusion medium, and having a gravity-based flow rate of
separation of less than 10 minutes to produce an eluted solution
comprising isolated nucleic acid; and c) collecting the eluted
solution for further analysis.
[0011] In an alternative embodiment, the present invention provides
a method of purifying a nucleic acid from a biological sample,
comprising the steps of: a) combining the biological sample with a
lysis buffer to form a lysis mixture comprising nucleic acid
released from cells in said biological sample; b) applying a volume
of the lysis mixture to size-exclusion chromatography medium in a
column comprising a loading end, an eluting end and a volume of
size-exclusion medium, optionally wherein said volume of lysis
mixture is 0.35 to 0.8 of the volume of the size-exclusion medium,
and allowing the lysis mixture to enter the size exclusion
chromatography medium; and c) providing a positive pressure
differential to the column to produce an eluted solution containing
nucleic acid for subsequent amplification or analysis. In certain
embodiments, the pressure differential is created by applying a
positive pressure to the loading side of the column. In another
embodiment the present invention provides a method of purifying
nucleic acids, comprising the steps of: a) applying a volume of a
sample containing nucleic acids to size-exclusion chromatography
medium in a column comprising a loading end, an eluting end, and a
volume of size-exclusion medium, and allowing sample to enter the
size-exclusion chromatography medium; and b) providing a negative
pressure differential or vacuum to the eluting end of the column
and collecting drained fluid containing nucleic acid for further
amplification or analysis.
[0012] The present invention further provides a method of purifying
a nucleic acid from a biological sample, comprising the steps of:
a) subjecting a volume of a mixture of biological sample containing
nucleic acid and a lysis agent to size-exclusion chromatography in
a column comprising a volume of size-exclusion medium, wherein said
volume of the mixture is 0.01 to 0.6 of the volume of the
size-exclusion medium, and having a gravity-based flow rate of
separation of less than 10 minutes to produce an eluted solution
comprising isolated nucleic acid; and c) collecting the eluted
solution for further analysis.
[0013] In certain embodiments, the invention provides further
equilibrating said chromatography column with an equilibrating
buffer comprising 1-10 mM Mg.sup.2+ or a non-ionic detergent, or a
combination, prior to the subjecting step that essentially will be
used for conducting the nucleic acid amplification step. In certain
embodiments, the invention provides that the lysis buffer comprises
alkali hydroxide at pH>11. In certain embodiments, the invention
provides that the lysis buffer comprises urea or chaotropic salts.
In certain embodiments, the invention provides that the lysis
buffer separates double stranded nucleic acid into single stranded
nucleic acid and inhibits nucleic acid interactions with
protein.
[0014] In certain embodiments, the invention provides that the
size-exclusion medium comprises a polymer such as polyacrylamide,
polybisacrylamide or polymethacrylamide. In certain embodiments,
the invention provides that the size-exclusion medium has a
molecular size exclusion limit of about 10 kDa or more. In certain
embodiments, the flow rate of separation is gravity-based.
[0015] The invention provides that the nucleic acid is either DNA,
RNA, or a mixture thereof. The invention further provides that the
amplifying step can be selected from the group consisting of
RT-PCR, qPCR, digital PCR, LAMP, sequencing, and an
enzyme-catalyzed reaction now known-or later developed. In certain
embodiments, the invention provides that the biological sample can
be selected from any source, including blood, saliva, stool, urine,
respiratory sample, or enriched culture broth.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 illustrates a universal sample preparation (USP)
workflow scheme.
[0017] FIG. 2 illustrates a universal sample preparation (USP)
workflow scheme using positive pressure.
[0018] FIG. 3 illustrates a universal sample preparation (USP)
workflow scheme using negative pressure.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides a universally applicable
nucleic acid purification, detection, and amplification method and
system using size-exclusion chromatography for separation of
nucleic acids from other cellular components. The method and system
provides isolated nucleic acids in less time than conventional
techniques in a form that is substantially free from other
inhibitors of nucleic acid amplification, detection, and/or
analysis. The isolated nucleic acid can also be prepared denatured
from a double-stranded to a single-stranded form during the
purification process, thus, eliminating a heat denaturing step in
the subsequent nucleic acid amplification and detection process.
These and other advantages and features of the invention will be
apparent to one of skill in the art.
[0020] In certain embodiments, the invention provides a method of
detecting a nucleic acid present in a biological sample, comprising
the steps of: a) combining the biological sample with a lysis
buffer to form a lysis mixture comprising nucleic acid released
from cells in said biological sample; b) subjecting a volume of the
lysis mixture to size-exclusion chromatography in a column
comprising a volume of size-exclusion medium, wherein said volume
of lysis mixture is 0.01 to 0.6 of the volume of the size-exclusion
medium, and having a flow rate of separation of less than about 10
minutes to produce an eluted solution comprising isolated nucleic
acid; c) combining the eluted solution with nucleic acid
amplification reagents comprising DNA polymerase, oligonucleotides
and nucleoside triphosphates for nucleic acid amplification; d)
amplifying the nucleic acid in the eluted solution; and e)
detecting products of nucleic acid amplification.
[0021] The present invention also provides a method of purifying a
nucleic acid from a biological sample, comprising the steps of: a)
combining the biological sample with a lysis buffer to form a lysis
mixture comprising nucleic acid released from cells in said
biological sample; and b) subjecting a volume of the lysis mixture
to size-exclusion chromatography in a column comprising a volume of
size-exclusion medium, wherein said volume of lysis mixture is 0.01
to 0.6 of the volume of the size-exclusion medium, and having a
flow rate of separation of less than about 10 minutes to produce an
eluted solution comprising isolated nucleic acid. In certain
embodiments, the invention provides the later step of optionally
amplifying and detecting or analyzing said isolated nucleic acid
using an enzyme-catalyzed reaction.
[0022] The present invention further provides a method of purifying
a nucleic acid from a biological sample, comprising the steps of:
a) combining the biological sample with a denaturing solution that
separates strands of double-stranded DNA; b) subjecting a volume of
the biological sample mixture to size-exclusion chromatography in a
column comprising a volume of size-exclusion medium, wherein
optionally said volume of lysis mixture is 0.01 to 0.6 of the
volume of the size-exclusion medium, and having a gravity-based
flow rate of separation of less than about 10 minutes to produce an
eluted solution comprising isolated nucleic acid; and c) collecting
the eluted solution for further amplification or analysis.
[0023] The present invention further provides a method of purifying
a nucleic acid from a biological sample, comprising the steps of:
a) subjecting a volume of a mixture of biological sample containing
nucleic acid and a lysis agent to size-exclusion chromatography in
a column comprising a volume of size-exclusion medium, wherein said
volume of the mixture is 0.01 to 0.6 of the volume of the
size-exclusion medium, and having a gravity-based flow rate of
separation of less than about 10 minutes to produce an eluted
solution comprising isolated nucleic acid; and b) collecting the
eluted solution for further amplification or analysis.
[0024] In practice, the method may be further modified by the use
of positive or negative pressure on the column. Positive pressure
can be applied to the loading end (top) of the column or negative
pressure can be applied to the eluting end (bottom) of the column.
When a pressure gradient is employed, optionally the volume of
lysis mixture can be 0.35 to 0.8 of the volume of the
size-exclusion medium.
[0025] In certain embodiments, the volume of the lysis mixture can
vary somewhat from between 0.01 to 0.6 of the volume of the
size-exclusion medium, to between 0.02 to 0.5 of the volume of the
size-exclusion medium, to between 0.1 to 0.4 of the volume of the
size-exclusion medium. In certain embodiments, the volume of the
lysis mixture is 200-400 .mu.l of sample per 800-1200 .mu.l of
filtration medium.
[0026] In certain embodiments, the invention provides a
gravity-based flow rate of separation of less than 10 minutes to
produce an eluted solution comprising isolated nucleic acid,
however, the rate can be less than 20 minutes, less than 15 minutes
or less than 12.5 minutes, and alternatively can be even less than
10 minutes at less than 9, 8, 7, 6, or 5 minutes.
[0027] In certain embodiments, the invention provides further
equilibrating said chromatography column with an equilibrating
buffer comprising 1-10 mM Mg.sup.2+ or a non-ionic detergent, or a
combination, prior to the subjecting step that essentially will be
used for conducting the nucleic acid amplification step.
[0028] In certain embodiments, the invention provides that the
size-exclusion medium comprises a polymer such as polyacrylamide,
polybisacrylamide or polymethacrylamide. In certain embodiments,
the invention provides that the size-exclusion medium has a
molecular size exclusion limit of about 10 kDa or more. In certain
embodiments, the flow rate of separation is gravity-based.
[0029] The invention provides that the nucleic acid is either DNA,
RNA, or a mixture thereof. The invention further provides that the
amplifying step can be selected from the group consisting of
RT-PCR, qPCR, digital PCR, LAMP, sequencing, and an
enzyme-catalyzed reaction now known or later developed. In certain
embodiments, the invention provides that the biological sample can
be selected from any source, including blood, saliva, stool, urine,
respiratory sample, or enriched culture broth.
[0030] In certain embodiments, the biological sample is mixed with
a lysis buffer, which causes disruption of cells or virus and
releases the nucleic acids. The lysis buffer used in the inventive
method comprises alkali hydroxide, such as NaOH or other alkalis,
with a pH more than 11, in combination with a detergent, such as
sodium dodecyl sulfate (SDS). Alternatively, the lysis buffer can
also comprise urea (for example 8M) or a chaotropic salt, such as
6M guanidinium salt. In certain embodiments, the invention provides
that the lysis buffer separates double stranded nucleic acid into
single stranded nucleic acid and inhibits nucleic acid interactions
with protein.
[0031] The present invention further provides that the biological
sample lysed with the lysis buffer is applied on the top of the SEC
gel filtration medium column and is allowed to enter the SEC
separating gel by gravity flow. After the sample solution enters
the gel medium completely, an additional volume of an elution
buffer can be applied on the top of the SEC gel filtration medium
and eluted solution in the last flow through is collected. In
certain embodiments, the elution buffer is molecular grade water.
In certain situations, it is advantageous to use water to move
nucleic acids through SEC gel filtration medium with a higher flow
rate.
[0032] In certain other embodiments, the equilibration and elution
buffer is a reaction buffer that is used for a subsequent
downstream reaction and/or application, including, but not limited
to, DNA/RNA detection and quantitation by various amplification and
detection methods, such as RT-PCR, qPCR, digital PCR, LAMP,
sequencing, and any other enzyme-catalyzed reactions. In certain
other embodiments, the elution buffer can be the same as the
equilibrating buffer, the separation buffer, and/or the reaction
buffer. In certain other embodiments, the elution buffer is
different from the equilibrating buffer, the separation buffer,
and/or the reaction buffer.
[0033] The present invention provides that because in certain
embodiments the equilibration buffer used in the inventive method
is substantially free from a nucleic acid amplification inhibitor
(such as endogenous protein or exogenous ethanol, or salts such as
guanadine chloride) that would interfere with nucleic acid
amplification and/or other downstream applications, the purified
nucleic acids obtained from the inventive method can be used for
various downstream applications discussed above. In certain
embodiments, the present invention provides that the elution buffer
is substantially free from a nucleic acid amplification
inhibitor.
[0034] A particular advantage of the inventive method is the
ability to rapidly obtain higher volumes of the purified nucleic
acid in the amplification reaction, thus, increasing the
sensitivity of detection, which is important for diagnostic
applications. The volume of eluted nucleic acid material can be
used in combination with a lyophilized pellet containing DNA
polymerase, dNTPs, and other components required for amplification
reaction.
[0035] The present invention further provides that in certain
embodiments the lysis buffer combined with the biological sample
can contain a denaturing agent to cause separation of
double-stranded DNA, resulting in single-stranded DNA during the
purification process. The examples of such agents include, but are
not limited to alkaline buffer at pH above 11 (0.01 M NaOH),
guanidinium salts (for example 6 M Guanidinium chloride), urea (for
example 8 M urea), and aqueous solutions of organic solvents (such
as formamide, dimethylformamide, or dimethylsulfoxide).
[0036] The inventive method thus provides purified nucleic acids
which can be used directly in the downstream nucleic acid
amplification methods without involving a DNA heat denaturation
step and additional equipment related thereto (such as a heating
block, or an instrument required to reach DNA-denaturation
temperature (usually 80-98.degree. C.), providing an advantage that
is particularly important for any isothermal amplification
technologies, including, but not limited to, loop mediated
amplification (LAMP), helicase dependent amplification (HAD),
recombinase amplification (RPA). In addition, separation of the
strands of DNA may result in change of the molecular shape of
nucleic acid molecules increasing apparent hydrodynamic size. This
effect may further limit the possibility of DNA entrance into the
pores of separating particles and increase DNA mobility in size
exclusion chromatography.
[0037] As used herein, "nucleic acids" refer to DNA, RNA or any
naturally occurring or synthetic modification thereof, and
combinations thereof. In certain embodiments, the nucleic acids are
DNA, which can be single, double or triple stranded or in any other
form, linear or circular. Nucleic acids that can be purified by the
method of current invention are polymers with minimal length of 10
bases and no limit on the maximal length. Particularly preferred
for isolation and detection in the present invention is genomic
DNA.
[0038] As used herein "isolated" means that a compound, such as a
nucleic acid, is separated from at least some of the constituents
with which it is associated in nature. "Isolated" and "purified"
may be used interchangeably herein.
[0039] As used herein, "biological samples" can be obtained from
materials from clinical samples for diagnosis, foods and allied
products, and environmental samples. In certain embodiments, such
biological samples can comprise all types of mammalian and
non-mammalian animal cells, plant cells and bacteria.
Representative samples include whole blood and blood-derived
products, such as plasma or buffy coat, saliva, semen, tissue
homogenates, urine, stool (feces), cerebrospinal fluid or any other
body fluids, tissues, cell cultures, cell suspensions, etc.
Biological material also includes environmental samples such as
soil, water, or food samples. The sample may also include
relatively pure or partially purified starting materials, such as
semi-pure preparations obtained by other cell separation
processes.
[0040] As used herein, "lysis buffer" refers to a buffered aqueous
solution, which breaks a cell wall or viral capsid, denatures
cellular or viral proteins and releases nucleic acids into said
solution. In certain embodiments, the lysis buffer includes sodium
hydroxide (NaOH) and sodium dodecylsulfate (SDS). In certain
embodiments, the lysis buffer comprises 0.01 to 2 M NaOH and 0.1 to
3% SDS. Alternatively, lysis buffer can contain chaotropic salts
(for example, Guanidine chloride), protein denaturants (for example
urea), buffers with extreme pH (more than 11) and detergents. In
certain embodiments, the lysis buffer may contain a nonionic
surfactant. The present invention contemplates or combination any
suitable surfactant that is determined by empirical selection and
evaluation, which is capable of lysing the cell membrane. Methods
of lysing cell using a lysis solution are well known in the art and
widely described in the literature. As mentioned above, some
embodiments of the lysis buffer can simultaneously cause lysis of
the cells or disruption of viral capsid and cause denaturation of
nucleic acids and separation of DNA strands. The lysis buffer can
be provided as a single solution or as separate solutions (for
example of alkali and detergent) which combined produce lysis
condition.
[0041] Conveniently, cell lysis can be achieved by using a lysis
buffer comprising chaotropes and/or detergents. For example, the
combination of a chaotrope with a detergent is found to be
particularly effective. An exemplary suitable lysis solution
includes a chaotrope, such as GTC or GHCl and a detergent, such as
SDS or Sarkosyl. The lysis agents are supplied in a simple aqueous
solution, or included in a buffer solution, to form a so-called
"lysis buffer". Any suitable buffer can be used, including for
example TRIS, BICINE, TRICINE and phosphate buffers. Alternatively,
the lysis agents are added separately. Suitable concentrations and
amounts of lysis agents vary according to the precise system and
can be appropriately determined.
[0042] As used herein, an "elution buffer" refers to a solution
that facilitates flow through a size-exclusion media column, and
can include the same composition as equilibrating buffer. In the
context of the size-exclusion chromatography, it is understood that
the main purpose of the elution buffer is to facilitate flow
through the column, and the elution buffer, if different from the
equilibration buffer, will reach the column exit only after nucleic
acids from the biological sample. In some embodiments the elution
buffer is a low viscosity solution at a pH which will not cause the
chemical decomposition of SEC medium. In some embodiments the
elution buffer is water. The elution buffer solution composition
may include a dye (for example Phenol Red dye) for easy visual
identification of the elution buffer.
[0043] As used herein, a "reaction buffer" refers to a buffered
solution that facilitates downstream amplification and detection,
and can include salts providing buffering capacity in the range of
pH optimal to conduct downstream amplification (examples include,
but are not limited to TRIS, BICINE, TRICINE), salts facilitating
or essential for downstream amplification reaction (such as
Mg.sup.2+ ions), salts creating optimal ionic strength to conduct
downstream amplification (including but not limited to NaCl, KCl),
detergents (such as TWEEN 20, TRITON X-100, etc) and potentially
other compounds which may increase performance of the downstream
nucleic acid amplification reaction. Some embodiments of the
reaction buffer composition are represented in the Examples
section.
[0044] As used herein, a "equilibration buffer" refers to a
buffered solution for packing SEC medium into a separating column
of the present invention. The composition of the "equilibration
buffer" can be the same composition as of the reaction buffer. In
other embodiments the composition of equilibration buffer is a
buffered solution providing stable storage of the purified nucleic
acid. The composition of the equilibration buffer in such
embodiments comprises buffering compound (examples include, but are
not limited to TRIS, BICINE, TRICINE) at pH within the range of pH
6-10. The composition of the equilibration buffer in such
embodiment can also include a metal-chelating compound (for example
EDTA) protecting nucleic acids from metal-mediated decomposition or
hydrolysis. The composition of the equilibration buffer in such
embodiments can also include a detergent (such as TWEEN 20, TRITON
X-100, etc) to prevent non-specific interaction of nucleic acid to
the SEC matrix. The composition of the equilibration buffer can
also include a carrier nucleic acid (such as yeast tRNA at 0.1 to
10 .mu.g/ml, polyA-polyT polymers, etc.) to prevent non-specific
interaction of nucleic acid to SEC matrix. The composition of the
equilibration buffer can also include inhibitors of bacterial
growth or disinfectants (for example sodium azide) to enable
ambient storage of packaged columns.
[0045] The present invention further provides that the biological
sample lysed with the lysis buffer is applied on the top of the SEC
gel filtration medium column and is allowed to enter the SEC
separating gel by gravity flow. The preferred scheme of the process
of this embodiment is presented in FIG. 1. In certain embodiments,
the SEC gel filtration medium column is equilibrated in
equilibrating buffer. In certain other embodiments, the SEC gel
filtration medium column is equilibrated in a reaction buffer for
downstream analysis (for example amplification). In certain other
embodiments, the biological sample lysed with the lysis buffer is
applied directly to the column without removing any of the
components in an intervening step between lysis and column
application.
[0046] Gel filtration or size exclusion chromatography is a method
in which molecules in solution are separated by their size. Small
molecules that can penetrate pores of the stationary phase can
enter the entire pore volume and the interparticle volume, and will
elute late. A very large molecule (such as a nucleic acid) that
cannot penetrate the pores moves in the interparticle volume
(.about.30-35% of the column volume) and will elute earlier when
this volume of mobile phase has passed through the column. The
underlying principle of SEC is that particles of different sizes
will elute (filter) through a stationary phase at different rates.
A preferred mode of gel filtration chromatography of the present
invention is using gravity flow, however, certain vacuum or
pressure modifications can be made to modify the flow rate as
described below.
[0047] Gel filtration medium is a stationary phase for gel
filtration chromatography. Particularly used for practicing of the
present invention are gel filtration mediums, which have pores
small enough to prevent nucleic acids from entering the pores, but
large enough to allow pore entrance by protein molecules, or other
organic (such as carbohydrates, lipids, etc) and inorganic
compounds and have minimal interaction with the surface of the
stationary phases. In certain embodiments, the SEC gel filtration
chromatography column has molecular size exclusion limit of about
10 kDa or more. More preferably, the SEC gel filtration mediums of
present invention include stationary phases which exclude molecules
with molecular weight more than 5.times.10.sup.6 Da, while
providing separation of molecules with molecular weight in the
range of 50,000 to 500,000 Da. The examples of gel filtration
medium useful for the present invention include, but are not
limited to: Sephacryls (S100, S200, S300, S400, S500, S1000),
Sepharoses (2B, 4B and 6), Toyopearls (HW-50, HW-55, HW-65, HW-75).
In certain other embodiments, the Sephacryl gel filtration medium
has a particle size of about 50 micrometers.
[0048] In certain embodiments, the SEC gel filtration medium is
packed into a disposable plastic column containing a porous filter
at the bottom. The SEC gel filtration medium is covered by another
porous filter and column can be capped at the top to prevent
evaporation for a long-term storage. In certain embodiments, the
design of the device utilizing the inventive method may include a
removable adsorbent pad, which can retain the liquid coming from
the SEC gel column during the sample application. In certain other
embodiments, the SEC gel-filtration medium is enclosed between
lower and upper porous filter disks having a porosity of about
40-100 micrometers.
[0049] After the sample solution enters the gel medium completely,
a volume of an elution buffer is applied on the top of the SEC gel
filtration medium to facilitate flow through the column.
Optionally, an additional volume of the elution buffer is applied
to provide more flow through the column. The volumes of the column,
the sample and the elution buffer can be selected to provide the
best separation with minimal dilution of applied nucleic acids.
Several examples of such ratios are presented in the Examples. The
volume displaced by the last volume of the elution buffer is
collected. Since the buffer applied to the SEC column after the
sample will always exit the column after the nucleic acids from the
sample, it's composition is determined mostly by convenience
factors. In certain embodiments, the elution buffer is molecular
grade water. It is advantageous to use water to move nucleic acids
through a SEC gel filtration medium with a higher flow rate. In
certain other embodiments, the elution buffer can be the same as
the equilibrating buffer, the separation buffer, and/or the
reaction buffer. In certain other embodiments, the elution buffer
is different from the equilibrating buffer, the separation buffer,
and/or the reaction buffer.
[0050] In an alternative embodiment of the invention, a lysis
mixture is applied on the column and is allowed to enter the
separation medium. A positive pressure differential is applied to
the column to produce an eluted solution containing nucleic acid.
The eluted solution containing nucleic acid is collected for
subsequent amplification or analysis. In certain embodiments,
positive pressure forces interstitial fluid from the SEC column,
thereby destroying the SEC column. In certain embodiments as
depicted in FIG. 2, the pressure differential is created by
applying a positive pressure to the loading side of the column. The
optimal ratio of the volume of lysis mixture to the volume of the
column is defined by two main factors: first the volume of the
lysis mixture should be larger than void volume of the column, and
second, the volume of the lysis mixture should be less than the
volume at which components of the lysis buffer would elute from the
column under constant flow conditions (without applying additional
pressure). In one embodiment, the volume of the lysis mixture is in
the range more than 0.35 volume of the SEC column and less than 0.8
volume of the SEC column (the volume of SEC volume is defined as
the volume of SEC medium packed into a column). In another
preferred embodiment, the volume of the lysis mixture is in the
range of more than 0.4 volume of the SEC column and less than 0.6
volume of the SEC column. In some embodiments of this invention,
the lysis mixture enters the column under gravity flow. Under
gravity flow conditions, flow spontaneously stops when the lysis
mixture enters the column preventing the column from draining.
[0051] The positive pressure differential can be provided by
different means, including, but not limited to, by means of
inserting a plunger or a pressurizing cap into a barrel of the SEC
column. The volume of the solution eluted from the column is
typically less than the expected void volume of the SEC column. In
the preferred embodiment this volume is in the range of 0.15-0.3
volume of the SEC column, and more preferably in the range of
0.175-0.25 volume of the SEC column.
[0052] Notably, the volume of collected liquid containing nucleic
acids is less than the volume of the applied lysis mixture. By
optimizing ratios of volume of SEC column, volume of lysis mixture
and volume of drained liquid, it is conceivable to achieve
conditions, under which nucleic acids are not diluted during said
SEC purification.
[0053] Moreover, if the leading front of the nucleic acid band
migrating through the column is slowed during migration due to
interaction with SEC medium (for example interaction of
electrostatic nature), is may be possible to obtain an effective
concentration of nucleic acid in the collected fraction.
[0054] In another alternative embodiment of the invention (depicted
in FIG. 3), a sample containing nucleic acids is applied on the
column and is allowed to enter the separation medium. A negative
pressure or vacuum is applied to the eluting or exit side of the
column forcing interstitial fluid containing nucleic acid to exit
the column. The eluted fluid containing nucleic acid is collected
for further amplification or analysis.
[0055] In certain embodiments, the ratio of the volume of the
sample to the SEC medium volume should be less than 1.0, and can be
in a range of 0.01 to 0.9. Optionally, an additional volume of
buffer could be applied to the column to move nucleic acids toward
the exit side of the column. The volume of the additional buffer is
typically less than the volume of the SEC medium in the column.
[0056] In some embodiments of this invention, the nucleic acid
sample enters the column under gravity flow. Under conditions of
gravity flow, the flow will spontaneously stop when the sample has
entered the SEC medium preventing the column from draining.
[0057] The negative pressure differential or vacuum can be provided
by different means, including, but not limited to, by means
connecting a tube (for example a Vacutainer tube) under a low
pressure to the exit side of the SEC column. The volume of the
interstitial liquid drained from the column is typically less than
the expected void volume of the SEC column. In the preferred
embodiment, this volume is in the range of 0.1-0.4 volume of the
SEC column, and in some embodiments in the range of 0.175-0.25
volume of the SEC column.
[0058] The negative pressure differential is the difference in the
pressure between pressure to which the SEC medium and eluting end
of the column are exposed. For example, if the SEC medium is under
atmospheric pressure conditions, the pressure applied to the
eluting end of the column is less than atmospheric pressure.
[0059] Notably, the volume of collected liquid containing nucleic
acids can be less than the volume of the applied sample containing
nucleic acids. By optimizing ratios of volume of SEC column, the
volume of lysis mixture and the volume of drained liquid, it is
possible to achieve conditions under which nucleic acids are not
diluted during said SEC purification.
[0060] Moreover, if the leading front of the nucleic acid band
migrating through the column is slowed during migration due to
interaction with SEC medium (for example electrostatic
interaction), it is possible to obtain an effective concentration
of nucleic acid in the collected fraction.
[0061] The alternative methods provide even faster separation,
being limited only by the time required for the lysis mixture to
enter the column bed formed by SEC medium.
[0062] In certain other embodiments, the equilibration buffer, in
which nucleic acids are eluted, is a reaction buffer that is used
for a subsequent downstream reaction and/or application, including,
but not limited to, DNA/RNA detection and quantitation by various
amplification and detection methods, such as RT-PCR, qPCR, digital
PCR, LAMP, sequencing, and any other enzyme-catalyzed reactions.
The equilibration buffer used in the present inventive method can
be substantially free from an inhibitor that would interfere with
nucleic acid amplification and/or other downstream
applications.
[0063] A particular advantage of the inventive method is the
ability to obtain a larger volume of the purified nucleic acid for
use in the amplification reaction, thus, increasing the sensitivity
of detection, which is particularly important for diagnostic
applications. The largest volume of eluted material can be used in
combination with a lyophilized pellet containing DNA polymerase,
dNTPs, and other component required for amplification reaction. As
used herein, the "DNA polymerase" is any enzyme that catalyzes or
helps to catalyze polymerization of deoxyribonucleotides into a DNA
strand using a nucleic acid (DNA or RNA) template. DNA polymerase
of present invention can be represented by a mixture of enzymes
with a DNA polymerase activity.
[0064] The present inventive method thus provides purified nucleic
acids to be used directly in the downstream nucleic acid
amplification methods without involving a DNA heat denaturation
step and additional equipment related thereto, such as a heating
block instrument required to reach DNA-denaturation temperature
(usually 80-98.degree. C.), providing an advantage that is
particularly important for isothermal amplification technologies,
including, but not limited to, loop mediated amplification (LAMP),
helicase dependent amplification (HAD), recombinase amplification
(RPA). As used herein, the "nucleic acid amplification" is the
process of increasing copy number of the original nucleic acid
sequence (DNA or RNA) using enzyme or enzymes catalyzing
polynucleotide synthesis. The examples of nucleic acid
amplifications of current invention include, but are not limited
to, polymerase chain reaction (PCR), loop-mediated amplification
(LAMP), template-mediated amplification (TMA).
[0065] The present invention further provides a kit for purifying
nucleic acid from a biological sample. In other embodiments a kit
is provided for detecting a nucleic acid present in a biological
sample. In certain embodiments, purification and detection kits may
include one or more of the following as herein described: a lysis
buffer; a size exclusion chromatography column; the components for
constructing a size exclusion chromatography column; tubes for
specimen, waste and/or sample collection; equilibration buffer.
Instructions for using the kits for nucleic acid purification
and/or detection according to the methods of the present invention
are preferably provided therewith.
[0066] Throughout this application, various publications are
referenced. The disclosures of all of these publications and those
references cited within those publications in their entireties are
hereby incorporated by reference into this application in order to
more fully describe the state of the art to which this invention
pertains.
[0067] It should also be understood that the foregoing relates to
preferred embodiments of the present invention and that numerous
changes may be made therein without departing from the scope of the
invention. The invention is further illustrated by the following
examples, which are not to be construed in any way as imposing
limitations upon the scope thereof. On the contrary, it is to be
clearly understood that resort may be had to various other
embodiments, modifications, and equivalents thereof, which, after
reading the description herein, may suggest themselves to those
skilled in the art without departing from the spirit of the present
invention and/or the scope of the appended claims.
[0068] Other features and advantages of the invention will be
apparent from the following description of the preferred
embodiments thereof and from the claims. These and many other
variations and embodiments of the invention will be apparent to one
of skill in the art upon a review of the appended description and
examples.
EXAMPLES
Example 1
Preparation of Separation Columns and USP Device
[0069] The USP device of the present example contains a plastic
column containing Sephacryl gel-filtration medium enclosed between
lower and upper porous filter disks. Gel volumes in a range of
0.83-1.32 ml were evaluated. A large number of columns with a
volume of SEC medium of 0.89 ml and 1.14 ml were prepared. Target
tolerance for gel volume +/-3%. The USP device was permanently
closed at the bottom prior to use. The user opened the fluid path
to initiate operation (by snapping off or cutting lower tip of the
column).
[0070] Fill Procedure: A lower disk was introduced in the present
inventive universal sample preparation (USP) device, and then
suspension of the Sephacryl gel (particle size 50 .mu.m) filtration
medium in Illumigene reaction buffer was dispensed and/or packed
into the device. The concentration of suspension was 40-60%, with
55% most frequently used. An upper disk was also introduced in the
device (40-100 .mu.m porosity of the filter disk allows buffer to
go through the filter, while locking gel particles between two
filters) to cover the Sephacryl gel filtration medium. The USP
Device was capped at the top to avoid evaporation. The USP device
with the Sephracryl gel filtration medium column can be stored at
2-27.degree. C. for prolonged periods of time (e.g., up to a year).
The USP device may include a removable adsorbent pad, which can
retain the liquid coming from the column during the sample
application.
Example 2
Viral RNA and Detection of Viruses
[0071] The Purpose of this experiment was to demonstrate the
ability of Universal Sample Preparation (USP) workflow to purify
viral RNA and the ability of Universal Sample Prep (USP) workflow
to detect viral nucleic acid. This experiment was done with
one-step RT reaction and amplification. The nasopharyngeal and
nasal clinical specimens were collected in transport medium. The
medium was frozen at -80.degree. C., until use. 200 ul of clinical
specimen was transport to a clean Eppendorf's tube. 50 .mu.l of
lysis buffer [0.2 N NaOH and 1% SDS) and 50 .mu.l of PBS buffer
supplemented with azide (PBSA) was added to same tube. The tube was
inverted 3 times to mix. 200 .mu.l of specimen mix was loaded onto
USP columns (equilibrated in LAMP reaction buffer {Trizma Base
4.8456 gm/ltr., KCl 1.4912 gm/ltr., Magnesium Sulfate, 2.5 M
(anhydrous) 6.4 ml/ltr., Ammonium Sulfate 2.6426 gm/ltr., Tween-20
2.0 ml/ltr., Sodium Azide 0.94 gm/ltr}).
[0072] Once specimen entered the gel, 200 ul of molecular grade
water was loaded on top of column. A clean elution tube was placed
for the final step. 200 ul of reaction buffer was added to the
column and eluted material was collected. 5 ul of elution was used
to perform one step RT-LAMP at 55.degree. C. The reaction was read
in real time on a Rotorgene Q real-time PCR thermocycler.
Composition of Florescent LAMP Reaction
[0073] Flu A florescent LAMP Reaction Compostion
TABLE-US-00001 dNTP's 1.4 mM 10% BSA, 0.6% FluA1 F3 primer 0.2 uM
FluA1 B3 primer 0.2 uM Flu AMFIP primer 1.6 uM FluA1 BIP primer 1.6
uM FluA1 LF primer 0.8 uM FAM FluA1 LB primer 0.5 uM BST
polymerase, 0.9 ul Reverse Transcriptase 1 U RNAse inhibitor (40
U/ul) 10 U
Flu B florescent LAMP Reaction Compostion
TABLE-US-00002 dNTP's 1.4 mM 10% BSA, 0.6% FluB1 F3 primer 0.2 uM
FluB1 B3 primer 0.2 uM FluB1FIP primer 1.6 uM FluB1 BIP primer 1.6
uM FluB1 LF primer 0.8 uM FB LBQ 0.04 uM BST polymerase, 0.9 ul
Reverse Transcriptase 1 U RNAse inhibitor (40 U/ul) 10 U
Flu A Specimen
TABLE-US-00003 [0074] ct Specimen ID value Detection PSH 1964 18.01
Positive PSH 0337 34.33 Positive PSH 1965 17.45 Positive PSH 1927
18.77 Positive PSH 1979 18.42 Positive
Flu B Specimen
TABLE-US-00004 [0075] ct Specimen ID value Detection PCMC 9410
20.76 Positive PCMC 4483 17.63 Positive PCMC 11207 14.38 Positive
PCMC 10457 15.32 Positive PCMC 11383 29.9 Positive
Influenza Negative Specimen
TABLE-US-00005 [0076] ct Specimen ID value Detection 7678 0
Negative 7679 0 Negative 7711 0 Negative 7756 0 Negative 7757 0
Negative
Example 3
Bacterial DNA and Detection in Stool Specimen
[0077] This experiment was performed to demonstrate the ability of
Universal Sample Prep workflow to lyse bacterial cells and to
obtain purified DNA. This experiment also demonstrates that USP
workflow purifies DNA from clinical stool specimen that is solid,
semi-solid and water. The purified DNA is ready for downstream
molecular applications.
[0078] Illumigene C. diff positive stool specimens were obtained
from clinical sites. The specimens were kept frozen at -80.degree.
C., until use. Consistency of stool specimens was observed to be
solid, semi-solid and water.
[0079] A full swab load of specimen was treated with 1480 ullysis
buffer {0.1 N NaOH and 0.54% SDS}. 50 ul of Assay control,
containing E. coli transformed with a plasmid encoding Spa gene of
Staphylococcus aureus. (Formaldehye fixed E. coli cells were
suspended in 10 mM Tris 0.1 mM EDTA at Absorbance 600 nm of
0.700-0.800. The cell suspension was diluted 1:10,000 in PBSA with
400 ng/ml yeast tRNA was added to the sample. Lysed sample was
vortexed to mix for 10 seconds and filtered through 7 .mu.m
filter.
[0080] 200 .mu.l of lysis mix was loaded onto USP columns made with
SEPHACRYLA S-300 and equilibrated in LAMP reaction buffer
(composition described above). Once specimen entered the gel, 200
.mu.l of molecular grade water was loaded on top of the column.
Once molecular grade water entered into gel completely, the waste
tube was removed from under the column. A clean elution tube was
placed for the final step. 200 .mu.l of molecular grade water was
added to the column and last flow through was collected in clean
elution tube. 50 .mu.l of elution was used to amplify toxin A gene
of C. diff in LAMP reaction at 63.degree. C. using Meridian C.
difficile test device (Catalog #280050). 50 .mu.l of elution was
use to amplify spa gene of S. aureus in LAMP reaction at 63.degree.
C.
[0081] Results of the amplification test are presented in a table
below:
TABLE-US-00006 Specimen Specimen Test Control Consistency ID (min)
(min) Solid #17 24 24 Solid #14 27 24 Solid #36 28 28 Semi-Solid
#48 26 23 Semi-Solid #42 22 25 Watery #19 23 27 Solid #17 24 24
Example 4
Urine Specimen
[0082] Columns were packed with 1.14 ml of Sephacryl S300 SEC
medium equilibrated in LAMP reaction buffer supplemented with yeast
tRNA as carrier. Leftover clinical urine samples, previously tested
for Chlamydia and Gonorrhea using nucleic acid amplification test
(ProbeTec (Beckton Dickinson)) were processed through USP
separation.
a) Urine Specimen Preparation
[0083] 1.5 mL of urine was transferred into a clean 2.0 mL micro
centrifuge tube and supplemented with a drop of Assay control
(diluted suspension of E. coli cells, harboring a plasmid
comprising sequence of Spa gene of Staphylococcus aureus, dispensed
from a dropper bottle. The tubes were centrifuged at 6000 rpm for 3
min. The supernatants were discarded by inverting tubes. Pellets
containing cells were resuspended in 250 .mu.L of lysis buffer (0.2
N NaOH and 1% SDS).
b) DNA Processing
[0084] 250 .mu.L of lysis mixture was applied to the USP column and
liquid was allowed to enter the column medium by gravity flow. 250
uL of buffer (containing Red dye to enhance visual tracking of
liquid flow through the top column filter) was applied to the
column and liquid was allowed to enter the column medium by gravity
flow. 250 .mu.L of elution buffer was applied to the column and
displaced buffer eluted from the column was collected into a clean
2.0 mL micro centrifuge tube.
c) LAMP Amplification:
[0085] 50 .mu.L of processed DNA sample were added to the TEST and
to the Control chambers of Illumigene devices (Meridian Bioscience,
Cincinnati, Ohio) containing lyophilized beads comprising dried
LAMP amplification reagents. A drop of Mineral oil was added to
each chamber to overlay reaction mixture. Illumigene devices were
closed and placed into illumiPro reader for 40 minute incubation at
63.degree. C. Optical signal was measured by the reader at the
beginning, during, and at the end of incubation. 55 out of 55
clinical urine samples positive for Chlamydia trahomatis by BD
ProbeTec were positive by LAMP amplification after processing
through USP. 13 out of 14 clinical urine samples positive for
Neisseria gonorrhea by BD ProbeTec were positive by LAMP
amplification after processing through USP. Average time to
positivity in test LAMP reactions was 20 minutes.
Example 5
Transport Media
[0086] The purpose of this experiment was to demonstrate the
feasibility of using Universal Sample Preparation workflow to lyse
cells and purify DNA from specimens that are collected in Transport
medium.
[0087] For this purpose, Purified Mycoplasma stock culture was
spiked into M4 Transport medium at 100 CFU/ml and 50 CFU/ml.
[0088] 300 ul of Mycoplasma sample was transferred to a clean tube.
50 .mu.l of assay control and 50 .mu.l of lysis buffer was added.
The mixture was inverted three times to mix. 350 ul of sample
mixture was loaded onto USP columns. The flow-through is collected
in a waste tube. Once the sample entered the column completely, the
waste tube was removed. A clean tube is placed under the column.
350 .mu.l of reaction buffer was added onto the column. The flow
through with purified DNA is collected in the clean tube.
[0089] 50 .mu.l of eluant is loaded into the test side of
Mycoplasma test devices to amplify Mycoplasma specific sequence. 50
.mu.l of eluant is loaded into the control side of Mycoplasma test
device to amplify spa specific sequence.
TABLE-US-00007 Mycoplasma Time for Amplification CFU/ml Test Avg
StdDev Detection 100 20.6 1.26 Positive 50 20 0 Positive
Example 6
Bronchoalveolar Lavage
[0090] This example presents evidence for Bronchoalveolar Lavage to
be a suitable clinical specimen for Mycoplasma DNA purification and
amplification with Universal Sample Prep workflow.
[0091] For this purpose appropriate dilution of Mycoplasma stock
culture was prepared in clinical matrix. The dilutions were 150
CFU/ml and 75 CFU/ml.
[0092] 200 ul of above dilutions was mixed with 50 .mu.l of lysis
buffer and 50 ul of assay control. Tube was inverted three times to
mix. 200 .mu.l of lysate was loaded onto USP column. Once the
entire sample entered the column, 200 .mu.l of molecular grade
water was loaded onto column. After collecting last drop of flow
through, the waste tube was discarded. A clean elution tube was
placed under USP column. 200 .mu.l of LAMP reaction buffer was
added to the column. The flow through was collected. 50 ul of
elution was added to Mycoplasma test device to test for presence of
Mycoplasma DNA. 50 ul of elution was added to control side of test
device to test the presence of control DNA
TABLE-US-00008 Samples Test Control 5 cfu/test FH Strain 1 at 23
min., 7 at 27 min., Mycoplasma pnuemoniae 7 at 25 min., 3 at 30
min. 1 at 30 min., 1 at 37 min., 2.5 cfu/test FH Strain 2 at 23
min., 3 at 27 min., Mycoplasma pnuemoniae 5 at 25 min., 7 at 30
min. 3 at 27 min.
Example 7
Blood Culture
[0093] This experiment was performed to demonstrate the feasibility
of amplifying DNA from blood and blood-related products with USP
workflow.
[0094] A blood culture that had been shown to be positive for
Staphylococcus aureus was tested using USP procedure. 400 ul of
positive blood culture was mixed with 100 .mu.l of lysis buffer.
The tube was inverted three times to mix. 200 .mu.l of lysate was
loaded onto USP column. 200 ul of 1.times. reaction buffer was
added to column followed by 200 ul of molecular grade water for
elution.
TABLE-US-00009 Time for Spa amplification # Specimen (min)
Detection 1 Blood culture 23 Positive 2 Blood culture 20
Positive
Example 8
Use of Sephacryl S-1000
[0095] The purpose of this experiment was to demonstrate the
feasibility of using Universal Sample Preparation workflow to lyse
cells and purify DNA from specimens that are collected in Transport
medium.
[0096] For this purpose, Purified Mycoplasma stock culture was
spiked into M4 Transport medium at 20 CFU/ml.
[0097] 300 .mu.l of Mycoplasma sample was transferred to a clean
tube. 50 ul of assay control and 50 .mu.l of lysis buffer was
added. The mixture was inverted three times to mix. 350 ul of
sample mixture was loaded onto USP columns. The flow-through is
collected in a waste tube. Once the sample entered the column
completely, the waste tube was removed. A clean tube is placed
under the column. 350 .mu.l of reaction buffer was added onto the
column. The flow through with purified DNA is collected in the
clean tube.
[0098] 50 .mu.l of eluant was loaded into the test side of
Mycoplasma test devices to amplify Mycoplasma specific sequence. 50
ul of eluant was loaded into the control side of Mycoplasma test
device to amplify spa specific sequence.
TABLE-US-00010 Myco Test Control CFU/ml (min) (min) 20 27 30 20 25
30 20 27 30 20 ND 27 20 30 30 20 30 30 20 ND 30 20 ND 30 20 27 30
20 ND 30
Example 9
Volume Ranges
[0099] This study was performed to demonstrate the versatility and
flexibility in volumes of sample, wash buffer and elution buffer in
USP workflow. Three different volumes of sample, wash and elution
buffer each were used in this study. C. diff spiked stool was used
as sample input and illumigene C. diff test was used the detect
DNA. C. diff spiked stool was diluted in pooled negative stool to a
final of 256 CFU/test.
[0100] A full swab load of specimen was mixed with 1480 .mu.l lysis
buffer. Two drops of Mycoplasma Assay control, containing E. coli
transformed with spa gene from Staphylococcus aureus was added to
sample. Sample was vortexed to mix for 10 seconds. 10 drops were
filtered through 7 .mu.m filter.
[0101] 160 .mu.l, 200 .mu.l and 240 .mu.l of sample were loaded
onto USP columns (made with SEPHACRYL S-300 and equilibrated in
LAMP reaction buffer). Once specimen entered the gel, 160 .mu.l,
200 .mu.ul and 240 .mu.l of molecular grade water was added. Once
molecular grade water entered into gel, completely, the waste tube
was removed from under the column. A clean elution tube was placed
for the final step. 160 .mu.l, 200 .mu.l and 240 .mu.l of molecular
grade water were added to the column and last flow through was
collected in clean elution tube. 50 .mu.l of elution was used to
amplify toxin A gene of C. diff in LAMP reaction at 63.degree. C.
50 ul of elution was use to amplify spa gene of S. aureus in LAMP
reaction at 63.degree. C.
TABLE-US-00011 Samples Volume Test Control 160 ul sample onto USP
(-20%) 7 at 27 min., 3 at 30 min. 10 at 23 min., 200 ul sample onto
USP 5 at 27 min., 5 at 30 min. 9 at 23 min., 1 at 25 min. 240 ul
sample onto USP (+20%) 3 at 25 min., 3 at 27 min., 10 at 23 min. 3
at 30 min., 1 at 33 min.
TABLE-US-00012 Wash Volume Test Control 160 ul Wash buffer 8 @ 30
min., 1 @ 40 min., 4 @ 23 min., (-20%) 1 ND 4 @ 25 min., 2 @ 27
min. Standard work flow 5 at 27 min., 5 at 30 min. 9 at 23 min., 1
at 25 min. 240 ul Wash buffer 1 @ 25 min., 6 @ 27 min., 9 @ 23
min., (+20% ) 1 @ 30 min., 2 ND 1 @ 30 min.,
TABLE-US-00013 Elution Volume Test Control 160 ul Elution 1 @ 25
min., 6 @ 27 min., 8 @ 23 min., (-20% ) 2 @ 30 min., 1 @ 40 min., 2
@ 25 min., Standard 5 at 27 min., 5 at 30 min. 9 at 23 min., work
flow 1 at 25 min. 240 ul Elution 1 @ 25 min., 5 @ 27 min., 10 @ 23
min., (+20%) 2 @ 30 min., 1 @ 33 min., 1 @ 35 min.,
Example 10
Testing of Volume Tolerances for USP
[0102] Determination of input and output volume tolerance of
extraction method is an important step in evaluating the efficacy
of the method. The following experiment was performed to test the
volume tolerances for USP. First columns were packed with 1.78 ml
(50% slurry of of Sephacryl S 300 hr made in 1.times. reaction
buffer). Mycoplasma pneumonia bacterial stock was diluted to 50
cfu/ml concentration in M4 medium and the internal control
containing E. coli cells harboring plasmid containing Spa gene of
Staphylococcus aureus was diluted to 10,000 times in PBSA buffer
with 400 ng/ml of yeast tRNA. Samples were prepared with varying
amounts of internal control, Mycoplasma and lysis buffer as shown
in the table to achieve the inputs of 250, 300 and 400 ul. Samples
were mixed 6 times gently with 200 ul automatic pippet. Prior to
the loading of sample to the columns, the excess buffer was drained
out. The applied samples were allowed to absorb into the gel
material for a minute and the flow thorough was discarded.
Following this process, nucleic acid was eluted using various
amounts (200, 250, 300 and 400 ul) of 1.times. reaction buffer. The
eluates were collected in clean 1.5 ml tubes for about one minute.
Fifty microliters of eluates were applied to Illumigene Mycoplasma
devices containing test and control beads. These devices were
further incubated at 63.degree. C. for 40 min in ILLUMIPRO-10
reader. Table shows that for a range of input and elution volumes,
the test and controls DNAs were detected.
TABLE-US-00014 Lysis Total Output Sample Control solution input
volume volume volume volume volume (.mu.l) Test Control (.mu.l)
(.mu.l) (.mu.l) (.mu.l) (elution) (min) (min) 100 50 100 250 300 33
37 200 50 100 400 300 30 33 150 50 100 300 250 30 33 100 100 100
300 400 30 33 100 50 150 300 200 33 33 200 50 150 400 200 30 33
Example 11
Sample Elution with a Syringe Plunger Instead of Gravity
[0103] Samples: Liquid Amies medium spiked with Chlamydia (CT) and
Gonorrhea (NG) cells as shown in the Table 1 below. One ml of the
Chlamydia and Gonorrhea spiked medium was mixed with approximately
40 .mu.l of assay control and 250 .mu.l of lysis buffer and mixed
the contents by simple inversions. M-Prep Columns: The M-prep
columns were packed with 1.14 ml of Sephacryl S300 hr slurry which
is saturated in illumigene reaction buffer. Five hundred micro
liters of the lysate loaded to the column and the eluted flow
through discarded. The nucleic acid sample was collected in a 1.5
ml tube from the columns with the help of a syringe plunger by
simply pushing the plunger inside the column. The eluted nucleic
acid volume obtained ranges from approximately 200 to 450 .mu.l.
illumigene Amplification: Fifty micro liters each of elute added to
the illumigene Chlamydia test and control reactions and another 50
.mu.l each of elute added to the illumigene Gonorrhea test and
control reactions. The loaded reaction devices incubated at 63 C
for 40 minutes on ILLUMIPRO-10 readers and collected the
amplification timings as shown in Table 1.
TABLE-US-00015 TABLE 1 Ten sample replicates tested at each
concentration. illumigene amplification detection times are shown
in minutes. Elution with plunger Liquid Amies Lysis Column Column
CT NG spiked with Sample Assay buffer input Output Test Control
Test Control CT and NG volume control volume volume volume (Time in
(Time in (Time in (Time in cells per ml (.mu.l) (.mu.l) (.mu.l)
(.mu.l) (.mu.l) minutes) minutes) minutes) minutes) 100 cfu/10 ifu
1000 40 250 500 200-450 9@20, 6@23, 7@20, 23, (n = 10) 23 2@25,
3@23 6@25, 27, 30 2@27, 33 30 cfu/3 ifu 1000 40 250 500 200-450
7@20, 4@23, 20, 2@23, (n = 10) 3@23 5@25, 4@23, 7@25, 35 4@25, 33
30 10 cfu/1 ifu 1000 40 250 500 200-450 2@20, 8@23, 20, 5@23, (n =
10) 5@23, 2@25 6@23, 5@25 25, 35, 25, ND 2@ND ND denotes no
detection.
Example 12
Sample Elution with the Sample Prep Device Cap Instead of Gravity
Combined With Detection by LAMP and PCR
[0104] Samples: Previously tested and Neisseria gonorrhea positive
frozen Urine samples selected for the study. One ml of the Urine
sample was mixed with approximately 40 .mu.l of assay control and
80 .mu.l of 1M NaOH followed by 250 .mu.l of lysis buffer and mixed
the contents by simple inversions.
[0105] M-Prep Columns: The M-prep columns were packed with 1.14 ml
of Sephacryl S300 hr slurry which is saturated in illumigene
reaction buffer. Five hundred or 700 micro liters of the lysate
loaded to the column and the eluted flow through discarded. The
nucleic acid sample was collected in a 1.5 ml tube from the columns
with the help of the M-prep column cap by simply pushing inside the
column. The eluted nucleic acid volume obtained was about 200-250
.mu.l.
[0106] illumigene Amplifications: Fifty micro liters each of elute
added to the illumigene Gonorrhea test and control reactions. The
loaded reaction devices incubated at 63.degree. C. for 40 minutes
on illumiPro-10 readers and collected the amplification timings as
shown in Table 2.
[0107] Real-Time PCR Amplifications: Five micro liters of the
eluted DNA from the each sample prep above mentioned method was
used in the PCR reactions. The reactions were setup using
Quantifast (Qiagen) master mix and the amplifications performed
using Rotor-Gene Q (Qiagen) thermal cycler. The Ct values are shown
in table 3. ND denotes that no amplification was noticed for those
particular samples
Example 13
Sample Elution Using Vacuumed Container Instead of Gravity Combined
with Detection by LAMP
[0108] Use of vacuum can be achieved by using a prepackaged
container with a closure that maintains a vacuum. A BD Vacutainer
was used to demonstrate the application. The SEC media utilized was
Sephacryl S-300 in 1.times.RB. The container used was a 10 ml
syringe with a luer lock fitting and a 26 gauge needle and filters
to keep the media in the container. The container was filled with
1.1 ml of SEC media.
[0109] The application experiment was done using contrived clinical
specimens at low concentrations. The clinical matrix was urine and
the target was Chlamydia cells. Assay Control was added to the
specimens, followed by the lysis buffer. Once mixed, 500 ul of
lysed specimen was added to the media and drained by gravity. Once
sample flow had stopped the target nucleic acid was collected by
puncturing a Vacutainer. The Vacutainer pulled the interstitial
fluid from the exterior of the media and left the internal fluid in
the Sephacryl bead. This material was then tested using illumigene
CT devices demonstrating successful amplification. See results in
Table 1.
TABLE-US-00016 TABLE 1 Results of Application Experiment illumigene
illumigene Contrived Test Result Control Result Specimen (n = 5) (n
= 5) CT (3 IFU) 5 Positive 5 Positive (3 @ 22, 2 @ 24) (4 @ 22, 1 @
24) CT (1 IFU) 5 Positive 5 Positive (4 @ 20, 1 @ 24) (2 @ 22, 3 @
24)
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